lithium-chloride and Leukemia

Fucoidan, a sulfated polysaccharide in brown seaweed, was found to inhibit proliferation and induce apoptosis in human lymphoma HS-Sultan cell lines. Fucoidan-induced apoptosis was accompanied by the activation of caspase-3 and was partially prevented by pretreatment with a pan-caspase inhibitor, z-VAD-FMK. The mitochondrial potential in HS-Sultan cells was decreased 24 hr after treatment with fucoidan, indicating that fucoidan induced apoptosis through a mitochondrial pathway. When HS-Sultan was treated with 100 microg/mL fucoidan for 24 hr, phosphorylation of ERK and GSK markedly decreased. In contrast, phosphorylation of p38 and Akt was not altered by treatment with fucoidan. L-selectin and P-selectin are known to be receptors of fucoidan; however, as HS-Sultan does not express either of these selectins, it is unlikely that fucoidan induced apoptosis through them in HS-Sultan. The neutralizing antibody, Dreg56, against human L-selectin did not prevent the inhibitory effect of fucoidan on the proliferation of IM9 and MOLT4 cells, both of which express L-selectin; thus it is possible fucoidan induced apoptosis though different receptors. These results demonstrate that fucoidan has direct anti-cancer effects on human HS-Sultan cells through caspase and ERK pathways.

Topics: Antibodies; Antineoplastic Agents; Apoptosis; Caspase 3; Caspases; Cell Line, Tumor; Down-Regulation; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Glycogen Synthase Kinases; Humans; L-Selectin; Leukemia; Lithium Chloride; Lymphoma; Multiple Myeloma; P-Selectin; Polysaccharides; Signal Transduction

To investigate the mechanism of proliferation inhibition and apoptosis of K562 leukemia cells by lithium chloride (LiCl), after K562 cells were treated with LiCl (30 mmol/L) cell cycle was examined by flow cytometry (FCM) and the expression of bcr/abl fusion gene mRNA was evaluated by RT-PCR. The intracellular Li(+) concentrations of K562 cells were determined at different time after treated with 30 mmol/L LiCl and the effects of TTX and FSK on intracellular Li(+) concentrations of K562 cells were also detected by atomic absorption spectrometry. The effects of TTX and FSK on LiCl-induced growth inhibition of K562 cells were determined by cell counting in liquid culture. The results showed that LiCl (30 mmol/L) caused a sustained arrest in G(2)/M cell cycle and down-regulated the bcr/abl mRNA expression in K562 cells, the intracellular Li(+) concentration of K562 cells increased at 30 minutes after treated with 30 mmol/L LiCl and reached apex at 2 hours, thereafter, gradually decreased and balanced at 4 hours after the treatment. If either Na(+) channel was pre-blocked with TTX or K(+) channel was pre-blocked with FSK, the intracellular Li(+) concentrations of K562 cells treated with 30 mmol/L LiCl were higher than that in the cells just treated with LiCl without pre-blocking. Furthermore, after pre-blocking either Na(+) channel with TTX or K(+) channel with FSK, the inhibition rate of K562 cell growth by 30 mmol/L LiCl could be increased. It is concluded that the mechanism of proliferation inhibition and apoptosis of K562 leukemia cells induced by LiCl is probably related with the G(2)/M cell cycle arrest, the bcr/abl mRNA expression down-regulation and the status of Na(+), K(+), or Li(+) ion channels on K562 leukemia cells.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Proliferation; Colforsin; Flow Cytometry; Fusion Proteins, bcr-abl; Gene Expression Regulation, Neoplastic; Humans; K562 Cells; Leukemia; Lithium Chloride; Potassium Channel Blockers; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Channel Blockers; Tetrodotoxin

We have previously shown that forced expression of the transcription factor Scl in WEHI-3B D(+) cells prevents ATRA-induced cell differentiation. We now find that the overexpression of Rbtn2 also interferes with induction of differentiation by ATRA. Addition of LiCl to ATRA treatment restored the capacity of both Scl- and Rbtn2-expressing cells to respond to the retinoid in a synergistic manner. Similar results were obtained with Scl-transfected HL60 cells where its expression diminished responsiveness to ATRA. These findings suggest that if Scl and/or Rbtn2 are involved in the non-responsiveness of AML patients to ATRA-induced differentiation, addition of LiCl may reverse insensitivity.

Topics: Adaptor Proteins, Signal Transducing; Adjuvants, Immunologic; Animals; Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Cell Differentiation; DNA-Binding Proteins; Drug Synergism; HL-60 Cells; Humans; Leukemia; LIM Domain Proteins; Lithium Chloride; Metalloproteins; Proto-Oncogene Proteins; T-Cell Acute Lymphocytic Leukemia Protein 1; Transcription Factors; Transfection; Tretinoin

LiCl interacts synergistically with all-trans-retinoic acid, promoting the terminal differentiation of WEHI-3B D(+) cells, a phenomenon partially due to the ability of the monovalent lithium cation to inhibit the proteasome-dependent degradation of retinoic acid receptor alpha protein. In this report, the 20S proteasome was purified from WEHI-3B D(+) cells and the effects of LiCl on chymotrypsin-like (Chtl) activity and peptidyl-glutamyl peptide hydrolyzing (PGPH) activity were determined. LiCl functions to inactivate both proteasomal activities in a time-dependent manner, without affecting non-proteasomal proteases. The half-lives for inactivation of Chtl and PGPH hydrolyzing activities were approximately 23 and 36min, respectively, at 10mM LiCl. Both SDS and peptide substrate increased the rate of inactivation. Partial enzymatic activity was recovered after dialysis in the absence of SDS, indicating that the off-rate for lithium was extremely slow. The findings suggest that the inactivation of Chtl and PGPH activities by LiCl occurs through a proteasomal conformational change.

Topics: Animals; Chymotrypsin; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Kinetics; Leukemia; Lithium Chloride; Multienzyme Complexes; Proteasome Endopeptidase Complex; Tumor Cells, Cultured

Lithium affects several enzymatic activities, however, the molecular mechanisms of lithium actions are not fully understood. We previously showed that LiCl interacts synergistically with all-trans-retinoic acid to promote terminal differentiation of WEHI-3B D(+) cells, a phenomenon accompanied by the recovery of the retinoid-induced loss of retinoic acid receptor alpha protein pools. Here, we demonstrate the effects of LiCl on proteasome-dependent degradation of retinoic acid receptor alpha proteins. LiCl alone, or in combination with all-trans-retinoic acid, increased cellular levels of ubiquitinated retinoic acid receptor alpha and markedly reduced chymotryptic-like activity of WEHI-3B D(+) 20 S and 26 S proteasome enzymes. Neither KCl nor all-trans-retinoic acid affected enzyme activity, whereas NaCl produced a modest reduction at relatively high concentrations. In addition, LiCl inhibited 20 S proteasome chymotryptic-like activity from rabbits but had no effect on tryptic-like activity of the 26 S proteasome. This effect has significant consequences in stabilizing the retinoic acid receptor alpha protein levels that are necessary to promote continued differentiation of leukemia cells in response to all-trans-retinoic acid. In support of this concept, combination of proteasome inhibitors beta-clastolactacystin or benzyloxycarbonyl-Leu-Leu-Phe with all-trans-retinoic acid increased differentiation of WEHI-3B D(+) cells in a manner that was analogous to the combination of LiCl and all-trans-retinoic acid.

Topics: Adjuvants, Immunologic; Animals; Blotting, Western; Cell Differentiation; Cell Line; Chymotrypsin; Cysteine Endopeptidases; Humans; Kinetics; Lactones; Leukemia; Lithium Chloride; Multienzyme Complexes; Oligopeptides; Peptide Hydrolases; Precipitin Tests; Proteasome Endopeptidase Complex; Protein Binding; Protein Biosynthesis; Rabbits; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Time Factors; Tretinoin; Trypsin; Tumor Cells, Cultured; Ubiquitin